Methods and compositions for secretion of heterologous polypeptides

ABSTRACT

The instant invention discloses the unexpected result that mutant signal sequences with reduced translational strength provided essentially complete processing and high levels of expression of a polypeptide of interest as compared to wild type signal sequences, and that many mammalian polypeptides require a narrow range of translation levels to achieve maximum secretion. A set of signal sequence vectors provides a range of translational strengths for optimizing expression of a polypeptide of interest.

FIELD OF THE INVENTION

This invention relates to signal sequences for the secretion of heterologous polypeptides from bacteria.

DESCRIPTION OF BACKGROUND AND RELATED ART

Secretion of heterologous polypeptides into the periplasmic space of E. coli and other prokaryotes or into their culture media is subject to a variety of parameters. Typically, vectors for secretion of a polypeptide of interest are engineered to position DNA encoding a secretory signal sequence 5′ to the DNA encoding the polypeptide of interest. Two major recurring problems plague the secretion of such polypeptides. First, the signal sequence is often incompletely processed or removed, and second, the amount of polypeptide secreted is often low or undetectable. Attempts to overcome these problems fall into three major areas: trying several different signal sequences, mutating the amino acid sequence of the signal sequence, and altering the secretory pathway within the host bacterium.

A number of signal sequences are available for the first approach in overcoming secretion problems. Watson (Nucleic Acids Research12: 5145-5164 (1984)) discloses a compilation of signal sequences. U.S. Pat. No. 4,963,495 discloses the expression and secretion of mature eukaryotic protein in the periplasmic space of a host organism using a prokaryotic secretion signal sequence DNA linked at its 3′ end to the 5′ end of the DNA encoding the mature protein. In particular, the DNA encoding E. coli enterotoxin signals, especially STII, are preferred. Chang et al. (Gene55:189-196 (1987)) discloses the use of the STII signal sequence to secrete hGH in E. coli. Gray et al. (Gene39:247-245 (1985)) disclose the use of the natural signal sequence of human growth hormone and the use of the E. coli alkaline phosphatase promoter and signal sequence for the secretion of human growth hormone in E. coli. Wong et al. (Gene68:193-203 (1988)) disclose the secretion of insulin-like growth factor 1 (IGF-1) fused to LamB and OmpF secretion leader sequences in E. coli, and the enhancement of processing efficiency of these signal sequences in the presence of a prlA4 mutation. Fujimoto et al. (J. Biotech.8:77-86 (1988)) disclose the use of four different E. coli enterotoxin signal sequences, STI, STII, LT-A, and LT-B for the secretion of human epidermal growth factor (hEGF) in E. coli. Denefle et al. (Gene85: 499-510 (1989)) disclose the use of OmpA and PhoA signal peptides for the secretion of mature human interleukin 1β.

Mutagenesis of the signal sequence has, in general, not been especially helpful in overcoming secretion problems. For example, Morioka-Fujimoto et al. (J. Biol. Chem.266:1728-1732 (1991)) disclose amino acid changes in the LTA signal sequence that increased the amount of human epidermal growth factor secreted in E. coli. Goldstein et al. (J. Bact.172:1225-1231 (1990)) disclose amino acid substitution in the hydrophobic region of OmpA effected secretion of nuclease A but not TEM β-lactamase. Matteucci et al. (Biotech.4:51-55 (1986)) disclose mutations in the signal sequence of human growth hormone that enhance secretion of hGH. Lehnhardt et al. (J. Biol. Chem.262:1716-1719 (1987) disclose the effect of deletion mutations in OmpA signal peptide on secretion of nuclease A and TEM β-lactamase.

Finally, attempts at improving heterologous secretion in E. coli by modulating host machinery has so far shown limited improvement in overcoming secretion problems. For example, van Dijl et al. (Mol. Gen. Genet.227:40-48 (1991)) disclose the effects of overproduction of the E. coli signal peptidase I (SPase I) on the processing of precursors. Klein et al. (Protein Engineering5:511-517 (1992) disclose that mutagenesis of the LamB signal sequence had little effect on secretion of bovine somatotropin, and that secretion properties of bovine somatotropin appear to be determined by the mature protein rather than by changes in the signal sequence. Perez-Perez et al. (Bio/Technology12:179-180 (1994)) disclose that providing an E. coli host with additional copies of prlA4 (secY allele) and secE genes, which encode the major components of the “translocator”, i.e., the molecular apparatus that physically moves proteins across the membrane, increased the ratio of mature to precursor hIL-6 from 1.2 to 10.8. U.S. Pat. No. 5,232,840 discloses novel ribosome binding sites useful in enhancing protein production in bacteria through enhanced and/or more efficient translation. U.S. Pat. No. 5,082,783 discloses improved secretion of heterologous proteins by hosts such as yeasts by using promoters of at most intermediate strength with heterologous DNA secretion signal sequences. European Patent Application No. 84308928.5, filed Dec. 19, 1984, discloses promoter-ribosome binding site expression elements of general utility for high level heterologous gene expression.

The instant invention discloses the unexpected result that altered translation initiation regions with reduced translational strength provided essentially complete processing and high levels of secretion of a polypeptide of interest as compared to wild type sequences, and that many mammalian polypeptides require a narrow range of translation levels to achieve maximum secretion. A set of vectors with variant translation initiation regions provides a range of translational strengths for optimizing secretion of a polypeptide of interest.

SUMMARY OF THE INVENTION

One aspect of the invention is a method of optimizing secretion of a heterologous polypeptide of interest in a cell comprising comparing the levels of expression of the polypeptide under control of a set of nucleic acid variants of a translation initiation region, wherein the set of variants represents a range of translational strengths, and determining the optimal translational strength for production of mature polypeptide, wherein the optimal translational strength is less than the translational strength of the wild-type translation initiation region.

In a further aspect of the invention the variants are signal sequence variants, especially variants of the STII signal sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the sequence of the PhoA promoter, Trp and STII Shine-Dalgarno regions and STII signal sequence.

FIG. 2 is a diagram depicting relevant features of the plasmid pLS33.

FIG. 3 is a diagram depicting construction of the library, pSTIIBK.

FIG. 4 is a graph depicting comparison of the levels of expression of IGF-1, as measured by the amount of IGF-1 detected in culture supernatants, for pLS33, pSTIIBK#131, and pSTIIC. Experiments 1 to 8 represent measurements taken on 8 separate dates.

FIG. 5 is a diagram depicting construction of the plasmid pSTIIC.

FIG. 6 is a diagram depicting construction of the plasmid pSTIILys.

FIG. 7 is a diagram depicting construction of the plasmid pPho21.

FIG. 8 is a diagram depicting construction of the plasmid pPho31.

FIG. 9 is a diagram depicting construction of the plasmid pPho41.

FIG. 10 is a diagram depicting construction of the plasmid pPho51.

FIG. 11 is a diagram depicting relevant features of the library, pSTIICBK.

FIG. 12 is a diagram depicting construction of the library, pSTBKPhoA.

FIG. 13 is a graph depicting PhoA activity in isolates of the pSTBKPhoA library.

FIG. 14 depicts the nucleotide sequences of the listed STII signal sequence variants.

FIG. 15 is a diagram depicting construction of the plasmid pNT3PST116.

FIG. 16 is a diagram depicting construction of the plasmid pST116Pho.

FIG. 17 is a diagram depicting relevant features of “category A” plasmids used in the examples.

FIG. 18 is a diagram depicting relevant features of “category B” plasmids used in the examples.

FIG. 19 is a photograph of a Coomassie blue stained polypeptide gel depicting secretion of mature ICAM-1 in E. coli under control of variant STII signal sequences. The TIR of relative strength 9 was provided by the pPho3 STII variant; the TIR of relative strength 3 was provided by the pPho41 STII variant. Precursor and mature forms of the polypeptide are indicated in the figure.

FIG. 20 is a photograph of a Coomassie blue stained polypeptide gel depicting secretion of mature NT3 in E. coli under control of variant STII signal sequences. The TIR of relative strength 9 was provided by the pPho31 STII variant; the TIR of relative strength 7 was provided by the pPho21 STII variant; the TIR of relative strength 3 was provided by the pPho41 STII variant; the TIR of relative strength 1 was provided by the pPho51 STII variant. The mature form of the polypeptide is indicated in the figure.

FIG. 21 is a photograph of a Coomassie blue stained polypeptide gel depicting secretion of mature RANTES in E. coli under control of variant STII signal sequences. Reading from left to right in the figure, the TIRs of relative strength 9 were provided by the pPho31and the pSTBKPhoA#116 STII variants; the TIR of relative strength 7 was provided by the pPho21 STII variant; the TIR of relative strength 4 was provided by the pSTBKPhoA#81 STII variant; the TIR of relative strength 3 was provided by the pPho41 STII variant, the TIR of relative strength 2 was provided by the pSTBKPhoA#107 STII variant; the TIRs of relative strength 1 were provided by the pSTBKPhoA#86 and the pPho51 STII variants. The mature form of the polypeptide is indicated in the figure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. Definitions

The “translation initiation region” or TIR, as used herein refers to a region of RNA (or its coding DNA) determining the site and efficiency of initiation of translation of a gene of interest. (See, for example, McCarthy et al. Trends in Genetics6:78-85 (1990).). A TIR for a particular gene can extend beyond the ribosome binding site (rbs) to include sequences 5′ and 3′ to the rbs. The rbs is defined to include, minimally, the Shine-Dalgarno region and the start codon, plus the bases in between, but can include the expanse of mRNA protected from ribonuclease digestion by bound ribosomes. Thus, a TIR can include an untranslated leader or the end of an upstream cistron, and thus a translational stop codon.

A “secretion signal sequence” or “signal sequence” as used herein refers to a sequence present at the amino terminus of a polypeptide that directs its translocation across a membrane. Typically, a precursor polypeptide is processed by cleavage of the signal sequence to generate mature polypeptide.

The term “translational strength” as used herein refers to a measurement of a secreted polypeptide in a control system wherein one or more variants of a TIR is used to direct secretion of a polypeptide encoded by a reporter gene and the results compared to the wild-type TIR or some other control under the same culture and assay conditions. For example, in these experiments translational strength is measured by using alkaline phosphatase as the reporter gene expressed under basal level control of the PhoA promoter, wherein secretion of the PhoA polypeptide is directed by variants of the STII signal sequence. The amount of mature alkaline phosphatase present in the host is a measure of the amount of polypeptide secreted, and can be quantitated relative to a negative control. Without being limited to any one theory, “translational strength” as used herein can thus include, for example, a measure of mRNA stability, efficiency of ribosome binding to the ribosome binding site, and mode of translocation across a membrane.

“Polypeptide” as used herein refers generally to peptides and polypeptides having at least about two amino acids.

B. General Methods

The instant invention demonstrates that translational strength is a critical factor in determining whether many heterologous polypeptides are secreted in significant quantities. Thus, for a given TIR, a series of amino acid or nucleic acid sequence variants can be created with a range of translational strengths, thereby providing a convenient means by which to adjust this factor for the optimal secretion of many different polypeptides. The use of a reporter gene expressed under the control of these variants, such as PhoA, provides a method to quantitate the relative translational strengths of different translation initiation regions. The variant or mutant TIRs can be provided in the background of a plasmid vector thereby providing a set of plasmids into which a gene of interest may be inserted and its expression measured, so as to establish an optimum range of translational strengths for maximal expression of mature polypeptide.

Thus, for example, signal sequences from any prokaryotic or eukaryotic organism may be used. Preferably, the signal sequence is STII, OmpA, PhoE, LamB, MBP, or PhoA.

Mutagenesis of the TIR is done by conventional techniques that result in codon changes which can alter the amino acid sequence, although silent changes in the nucleotide sequence are preferred. Alterations in the TIR can include, for example, alterations in the number or spacing of Shine-Dalgarno sequences, along with alterations in the signal sequence. One preferred method for generating mutant signal sequences is the generation of a “codon bank” at the beginning of a coding sequence that does not change the amino acid sequence of the signal sequence (i.e., the changes are silent). This can be accomplished by changing the third nucleotide position of each codon; additionally, some amino acids, such as leucine, serine, and arginine, have multiple first and second positions that can add complexity in making the bank. This method of mutagenesis is described in detail in Yansura et al. (METHODS: A Companion to Methods in Enzymol.4:151-158 (1992)). Basically, a DNA fragment encoding the signal sequence and the beginning of the mature polypeptide is synthesized such that the third (and, possibly, the first and second, as described above) position of each of the first 6 to 12 codons is altered. The additional nucleotides downstream of these codons provide a site for the binding of a complementary primer used in making the bottom strand. Treatment of the top coding strand and the bottom strand primer with DNA polymerase I (Klenow) will result in a set of duplex DNA fragments containing randomized codons. The primers are designed to contain useful cloning sites that can then be used to insert the DNA fragments in an appropriate vector, thereby allowing amplification of the codon bank. Alternative methods include, for example, replacement of the entire rbs with random nucleotides (Wilson et al., BioTechniques17:944-952 (1994)), and the use of phage display libraries (see, for example, Barbas et al., Proc. Natl. Acad. Sci. U.S.A.89:4457-4461 (1992); Garrard et al., Gene128:103-109 (1993)).

Typically, the TIR variants will be provided in a plasmid vector with appropriate elements for expression of a gene of interest. For example, a typical construct will contain a promoter 5′ to the signal sequence, a restriction enzyme recognition site 3′ to the signal sequence for insertion of a gene of interest or a reporter gene, and a selectable marker, such as a drug resistance marker, for selection and/or maintenance of bacteria transformed with the resulting plasmids.

Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems (Chang et al., Nature275:617-624 (1978); and Goeddel et al., Nature281:544-548 (1979)), alkaline phosphatase, a tryptophan (Trp) promoter system (Goeddel, Nucleic Acids Res.8(18):4057-4074 (1980) and EP 36,776) and hybrid promoters such as the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. U.S.A.80:2114 25 (1983).

Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem.255(24):12073-80 (1980)) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg.7:149-67 (1968)); and Holland, Biochemistry17:4900-4907 (1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phospho-fructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in Hitzeman et al., EP 73,657A. Yeast enhancers also are advantageously used with yeast promoters.

Any reporter gene may be used which can be quantified in some manner. Thus, for example, alkaline phosphatase production can be quantitated as a measure of the secreted level of the phoA gene product. Other examples include, for example, the β-lactamase genes.

Preferably, a set of vectors is generated with a range of translational strengths into which DNA encoding a polypeptide of interest may be inserted. This limited set provides a comparison of secreted levels of polypeptides. The secreted level of polypeptides can be determined, for example, by a functional assays for the polypeptide of interest, if available, radioimmunoassays (RIA), enzyme-linked immunoassays (ELISA), or by PAGE and visualization of the correct molecular weight of the polypeptide of interest. Vectors so constructed can be used to transform an appropriate host. Preferably, the host is a prokaryotic host. More preferably, the host is E. coli.

Further details of the invention can be found in the following examples, which further define the scope of the invention. All references cited herein are expressly incorporated by reference in their entirety.

EXAMPLES

I. Plasmid Constucts

A. Basic Plasmid Construction

All of the plasmids described in this patent application were constructed from a basic backbone of pBR322 (Sutcliffe, Cold Spring Harb Symp Quant Biol.43:77-90 (1978)). While the gene of interest expressed in each case varies, the transcriptional and translational sequences required for the expression of each gene were provided by the PhoA promoter and the Trp Shine-Dalgarno sequence (Chang et al., Gene55:189-196 (1987)). Additionally, in the cases noted, a second Shine-Dalgarno sequence, the STII Shine-Dalgarno sequence (Picken et al., Infect Immun42(1):269-275 (1983)), was also be present. Secretion of the polypeptide was directed by the STII signal sequence or variants thereof (Picken et al., Infect Immun42(1):269-275 (1983)). The PhoA promoter, Trp and STII Shine-Dalgarno sequences and the sequence of the wild-type STII signal sequence are given in FIG. 1.

B. Construction of pLS33

The plasmid pLS33 was derived from phGH1 (Chang et al., Gene55:189-196 (1987)), which was constructed for the expression of des(1,3)-IGF-I. In the plasmid pLS33, the gene encoding this version of insulin-like growth factor I (altered from the original sequence (Elmblad et al., Third European Congress on Biotechnology III. Weinheim: Verlag Chemie, pp.287-292 (1984)) by the removal of the first three amino acids at the N-terminus) replaced the gene encoding human growth hormone. The construction pLS33 maintained the sequences for the PhoA promoter, Trp and STII Shine-Dalgarno regions and the wild-type STII signal sequence described for phGH1. However, the 3′ end following the termination codon for des(1, 3)-IGF-I was altered from that described for phGH1. In the case of pLS33, immediately downstream of the termination codon a HindIII restriction site was engineered, followed by the methionine start codon of the tetracycline resistance gene of pBR322 (Sutcliffe, Cold Spring Harb Symp Quant Biol43:77-90 (1978)). A diagram of the plasmid pLS33 is given in FIG. 2.

C. Construction of pSTIIBK

A plasmid library containing a variable codon bank of the STII signal sequence (pSTIIBK) was constructed to screen for improved nucleotide sequences of this signal. The vector fragment for the construction of pSTIIBK was created by isolating the largest fragment when pLS33 was digested with XbaI and BstEII. This vector fragment contains the sequences that encode the PhoA promoter, Trp Shine-Dalgarno sequence and amino acids 16-67 of des(1,3)-IGF-I. The coding region for amino acids 3-15 of des(1,3)-IGF-I was provided by isolating the DraIII - BstEII fragment (approximately 45 bp) from another IGF-I expression plasmid, pLS331amB. The variations in the nucleotide sequence for the STII signal were derived from the two strands of synthetic DNA listed below:

5′ - GCATGTCTAGAATT ATG AAR AAR AAY ATH GCN TTY CTN CTN GCN TCN ATG TTY GTN TTY TCN ATH GCT ACA AAC GCG TAT GCC ACTCT -3′ (SEQ ID NO:1)             3′- CGA TGT TTG CGC ATA CGG TGAGACACGCCACGACTT - 5′ (SEQ ID NO:2) R: A, G Y: T, C H: A, T, C N: G, A, T, C

These two strands of synthetic DNA were annealed and treated with DNA Polymerase I (Klenow Fragment) to form duplex DNA of approximately 101 bp. This duplex DNA was then digested with XbaI and DraIII to generate the fragment of approximately 82 bp encoding the STII signal sequence with variable codons and the first two amino acids of des(1,3)-IGF-I. These fragments were then ligated together as shown in FIG. 3 to construct the library, pSTIIBK.

D. Selection of pSTIIBK#131

The plasmid library containing a variable codon bank of the STII signal sequence (pSTIIBK) was screened for improved growth of transformants and increased secretion of IGF-1. Basically, plasmids were transformed into host strain 27C7 (see below) and screened for enhanced ability to grow in a low phosphate medium (see Chang et al., supra) plus carbenicillin (50 μg/ml) based on OD₆₀₀ measurements of cell density. Candidate colonies were tested for increased levels of IGF-1 secretion as follows. Colonies were inoculated into 3-5 ml LB plus carbenicillin (50 μg/ml) and grown at 37° C. with shaking for about 5-15 hours. Cultures were diluted 1:100 into 1-3 ml low phosphate medium plus Carbenicillin (50 μg/ml) and induced for 24 hours shaking at 37° C. The induced cultures were centrifuged in microcentrifuge tubes for 5 minutes. Supernatants were diluted into IGF RIA diluent and stored at −20° C. until assayed. The amount of IGF-1 secreted into the medium was measured by a radioimmunoassay.

The level of expression of IGF-1, as measured by the amount of IGF-1 detected in culture supernatants, was compared for pLS33, pSTIIBK#131, and pSTIIC, in FIG. 4. The variant #131 consistently improved IGF-1 expression over the “original”, or wild-type STII signal sequence. pSTIIC showed some slight improvement in expression over the wild-type sequence. pSTIIBK#131 differed from the wild-type STII in 12 codons and in the deletion of one Shine-Dalgarno sequence. pSTIIC was constructed as described below as a control plasmid having only one Shine-Dalgarno sequence and three codon changes near the extreme 3′ end of the signal.

E. Construction of pSTIIC

In pSTIIC the STII Shine-Dalgarno sequence was removed from the plasmid pLS33. In addition, by incorporating silent mutations near the 3′ end of the STII signal, an MluI site was engineered into pSTIIC. The identical fragments described for the construction of pSTIIBK (the vector from pLS33 and the approximately 45 bp DraIII - BstEII fragment from pLS331amB) were used for the construction of this plasmid. However, the synthetic DNA differed from that described above for the construction of pSTIIBK. For the construction of pSTIIC, the synthetic DNA coding for the STII signal sequence and the first two amino acids of des(1,3)-IGF-I was as follows:

5′ - CTAGAATT ATG AAA AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT     3′- TTAA TAC TTT TTC TTA TAG CGT AAA GAA GAA CGT AGA TAC AAG CAA                      __MluI__ TTT TCT ATT GCT ACA AAC GCG TAT GCC ACTCT - 3′ (SEQ ID NO:3) AAA AGA TAA CGA TGT TTG CGC ATA CGG TG - 5′ (SEQ ID NO:4)

These fragments were ligated together as illustrated in FIG. 5 to construct the plasmid pSTIIC.

F. Construction of pSTILys

The plasmid pSTIILys contained an STII signal sequence that differs from the signal sequence of pSTIIC by only one nucleotide change at the position of the second codon. This signal sequence was constructed from synthetic DNA and placed in a pBR322-based vector for the expression of the polypeptide RANTES (Schall et al., J Immunol141(3):1018-1025 (1988)). The XbaI - MluI vector fragment for this construction was isolated from the plasmid pBK131Ran (a derivative of the plasmid pSTIIBK#131 with the gene encoding RANTES replacing the gene encoding des(1,3)-IGF-I). This vector contained the PhoA promoter, Trp Shine-Dalgarno sequence, the last three amino acids of the STIIC signal sequence and the gene encoding the polypeptide RANTES. As illustrated in FIG. 6, this fragment was then ligated with the following strands of synthetic DNA to construct the plasmid pSTIILys (SEQ ID NO:3):

5′- CTAGAATT ATG AAG AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT      3′-TTAA TAC TTC TTC TTA TAG CGT AAA GAA GAA CGT AGA TAC AAG CAA TTT TCT ATT GCT ACA AA - 3′ (SEQ ID NO:5) AAA AGA TAA CGA TGT TTG CGC - 5′ (SEQ ID NO:6)

G. Construction of Alkaline Phosphatase Plasmids

In order to determine a quantitative TIR value for each of the STII signal sequences described, the alkaline phosphatase gene of E.coli was used as a reporter gene. In each of these constructions, the PhoA gene was placed downstream of the PhoA promoter, Trp Shine-Dalgarno sequence and a version of the STII signal sequence. The plasmids pPho21, pPho31, pPho41 and pPho51 contained the signal sequences derived from pSTIIC, pLS33, pSTIIBK#131 and pSTIILys, respectively. In the case of pPho3, the construction also contained he STII Shine-Dalgarno region.

H. Construction of pPho21

The vector fragment for the construction of pPho2l was created by digesting pBR322 with EcoRI and BamHI and isolating the largest fragment. The PhoA promoter, Trp Shine-Dalgarno sequence and STIIC signal sequence (amino acids 1-20) were provided by isolating the approximately 484 bp fragment of pCN131Tsc following digestion with EcoRI and MluI. An identical fragment of approximately 484 bp could have also been generated from pSTIIC, a plasmid which has been described previously. The PhoA gene fragment (approximately 1430 bp) encoding amino acids 24-450 of alkaline phosphatase was generated from the plasmid pbO525 following digestion with Bsp1286 and BamHI (Inouye et al., J Bacteriol146(2):668-675 (1981)). This Bsp1286 - BamHI fragment also contains approximately 142 bp of SV40 DNA (Fiers et al., Nature273:113-120 (1978)) following the termination codon of alkaline phosphatase. Synthetic DNA was used to link the STII signal sequence with the PhoA gene. The sequence of this DNA encoding the last three amino acids of the STIIC signal sequence and amino acids 1-23 of alkaline phosphatase was as follows:

5′ - CGCGTATGCCCGGACACCAGAAATGCCTGTTCTGGAAAACCGGGCTGCTCAGGGCGATATTACTG     3′ - ATACGGGCCTGTGGTCTTTACGGACAAGACCTTTTGGCCCGACGAGTCCCGCTATAATGAC CACCCGGCGGTGCT - 3′ (SEQ ID NO:7) GTGGGCCGCC - 5′ (SEQ ID NO:8)

In order to facilitate the construction of this plasmid, the synthetic DNA was preligated to the EcoRI-MluI fragment of pCN131Tsc. This preligation generated a new fragment of about 575 bp. As illustrated in FIG. 7, the fragment generated from the preligation was then ligated together with the other fragments described to construct pPho2.

I. Construction of pPho31

The vector fragment for the construction of this plasmid was the identical vector described for pPho2. The PhoA promoter, Trp Shine-Dalgarno sequence, STII Shine-Dalgarno sequence and STII signal sequence (amino acids 1-20) were generated from pJAL55. The necessary fragment (approximately 496 bp) from pJAL55 was isolated following digestion with EcoRI and MluI. This EcoRI-MluI fragment only differed from the same region of pLS33 by an engineered MluI site starting at amino acid 20 of the STII signal sequence (as described for pSTIIC). The last three amino acids of the STIIC signal sequence and the sequence encoding the PhoA gene were provided by digesting the plasmid pPho21 with MluI and BamHI and isolating the approximately 1505 bp fragment. These fragments were ligated together as shown in FIG. 8 to yield pPho31.

J. Construction of pPho41

The vector fragment for the construction of this plasmid was the identical vector described for pPho21. The PhoA promoter, Trp Shine-Dalgarno sequence and STII signal sequence with pSTIIBK#131 codons (amino acids 1-20 ) were provided by isolating the approximately 484 bp EcoRI-MluI fragment of pNGF131. An identical fragment could have also been generated from pSTIIBK#131. The last three amino acids of the STIIC signal sequence and the sequence encoding the PhoA gene were provided by digesting the plasmid pPho21 with MluI and BamHI and isolating the approximately 1505 bp fragment. As illustrated in FIG. 9, these three fragments were then ligated together to construct pPho41.

K. Construction of pPho51

The vector fragment for the construction of pPho51 was generated by digesting the plasmid pLS18 with XbaI-BamHI and isolating the largest fragment. The plasmid pLS18 is a derivative of phGH1 (Chang et al., Gene55:189-196 (1987)) and an identical vector would have been generated had phGH1 been used in place of pLS18. This XbaI-BamHI vector contains the PhoA promoter and the Trp Shine-Dalgarno sequence. The STII signal sequence (amino acids 1-20) with pSTIILys codons was provided by isolating the approximately 67 bp fragment generated when pSTIILys was digested with XbaI and MluI. The last three amino acids of the STIIC signal sequence and the sequence encoding the PhoA gene were provided by digesting the plasmid pPho21 with MluI and BamHI and isolating the approximately 1505 bp fragment. A diagram for the construction of pPho51 is given in FIG. 10.

L. Construction of pSTIICBK

A second variable codon library of the STII signal sequence, pSTIICBK, was constructed. This second codon library was designed only to focus on the codons closest to the met initiation codon of the STII signal sequence. As illustrated in FIG. 11, pSTIICBK was a pBR322-based plasmid containing the gene encoding the polypeptide RANTES (Schall et al., J Immunol141(3):1018-1025 (1988)) under the control of the PhoA promoter and the Trp Shine-Dalgarno sequence. In this plasmid, secretion of RANTES is directed by an STII signal sequence codon library derived from the following two strands of synthetic DNA:

5′- GCATGTCTAGAATT ATG AAR AAR AAY ATH GCN TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCC-3′ (SEQ ID NO:9)     3′- AGA TAA CGA TGT TTG CGC ATA CGG TGA - 5′ (SEQ ID NO:10) R: A, G Y: T, C H: A, T, C N: G, A, T, C

These two strands of synthetic DNA were annealed and treated with DNA Polymerase I (Klenow Fragment) to form duplex DNA of approximately 86 bp. This duplex DNA was then digested with XbaI and MluI to generate a fragment of approximately 67 bp encoding the first 20 amino acids of the STII signal sequence with variable codons at positions 2-6.

M. Construction of pSTBKPhoA

To increase the number of STII signal sequences available with differing relative TIR strengths, a convenient method of screening the codon library of pSTIICBK was required. The plasmid pSTBKPhoA was constructed as a solution to this problem. In the plasmid pSTBKPhoA, the STII codon library of pSTIICBK was inserted upstream of the PhoA gene and downstream of the PhoA promoter and the Trp Shine-Dalgarno sequence. PhoA activity thus provided a means by which to discriminate between different versions of the STII signal sequences.

The vector fragment for this construction was created by isolating the largest fragment when p131TGF was digested with XbaI and BamHI. An identical vector could have also been generated from phGH1 (Chang et al., Gene55:189-196 (1987)). This vector contained the PhoA promoter and the Trp Shine-Dalgarno sequence. The codon library of the STII signal sequence was provided by isolating the approximately 67 bp fragment generated from pSTIICBK following digestion with XbaI and MluI. The last three amino acids of the STIIC signal sequence and the sequence encoding the PhoA gene were provided by digesting pPho2 with MluI and BamHI and isolating the approximately 1505 bp fragment. As illustrated in FIG. 12, the fragments were then ligated together to construct pSTBKPhoA.

N. Selection of pSTBKPhoA #81, 86, 107, 116

The plasmids pSTBKPhoA #81, 86, 107, 116 were selected from the codon library of pSTBKPhoA based on their basal level PhoA activity (FIG. 13). As listed in FIG. 14, each had a different nucleotide sequence encoding the STII signal sequence.

O. Construction of pST116Pho

This version of the STII signal sequence, ST116, combined the double Shine-Dalgarno sequence described by Chang et al. (Gene 55:189-196 (1987)) with the codons of the selected STII sequence pSTBKPhoA #116. This signal sequence was initially constructed in a plasmid designed for the secretion of the pro region of NT3 (pNT3PST116) and then was transferred into a plasmid containing the PhoA gene to obtain a relative TIR measurement (pST116Pho).

P. Construction of pNT3PST116

The vector for this construction was generated by digesting the plasmid pLS18 with XbaI and BamHI and isolating the largest fragment. The plasmid pLS18 was a derivative of phGH1 (Chang et al., Gene55:189-196 (1987)) and an identical vector could have been generated from phGH1. This XbaI-BamHI vector contained the PhoA promoter and the Trp Shine-Dalgarno sequence. A fragment (approximately 682 bp) containing the last three amino acids of the STII signal sequence and the coding region for amino acids 19-138 of proNT3 (Jones et al., Proc Natl Acad Sci 87:8060-8064 (1990)) was generated from the plasmid pNT3P following digestion with MluI and BamHI. The plasmid pNT3P was a pBR322-based plasmid containing the PhoA promoter, STIIBK#131 version of the STII signal sequence and the coding region for amino acids 19-138 of proNT3. The strands of synthetic DNA listed below provided the sequence for the STII Shine-Dalgarno sequence and the first 20 amino acids of the STII signal sequence:

5′ - CTAGAGGTTGAGGTGATTTT ATG AAA AAA AAC ATC GCA TTT CTT CTT GCA TCT     3′ - TCCAACTCCACTAAAA TAC TTT TTT TTG TAG CGT AAA GAA GAA CGT AGA ATG TTC GTT TTT TCT ATT GCT ACA AA - 3′ (SEQ ID NO:11) TAC AAG CAA AAA AGA TAA CGA TGT TTG CGC - 5′ (SEQ ID NO:12)

These fragments were then ligated together as shown in FIG. 15 to construct pNT3PST116.

Q. Construction of ST116Pho

The vector for the construction of this plasmid was the identical vector described for the construction of pNT3PST116. The STII Shine-Dalgarno sequence and the first 20 amino acids of the STII signal sequence (pSTBKPhoA#116 codons) were generated by isolating the approximately 79 bp fragment from pNT3PST116 following digestion with XbaI and MluI. The last three amino acids of the STIIC signal sequence and the sequence encoding the PhoA gene were isolated from pSTBKPhoA#116 following digestion with MluI and BamHI (approximately 1505 bp fragment). As illustrated in FIG. 16, ligation of these three fragments resulted in the construction of pST116Pho.

II. Alkaline Phosphatase Assay

In these experiments the altered TIR constructs utilizing the phoA reporter gene were assayed for relative translational strengths by a modification of the method of Amenura et al. (J. Bacteriol.152:692-701, 1982).

Basically, the method used was as follows. Plasmids carrying altered sequences, whether in the TIR, the Shine-Dalgarno region, the nucleotide sequence between the Shine Dalgarno region and the start codon of the signal sequence, or the signal sequence itself, whether amino acid sequence variants or nucleotide sequence variants, were used to transform E. coli strain 27C7 (ATCC 55,244) although any PhoA- strain of E. coli could be used. Transformant colonies were inoculated into Luria-Bertani medium (LB) plus carbenicillin (50 μg/ml, Sigma, Inc.). Cultures were grown at 37° C. with shaking for 4-8 hr. The equivalent of 1 OD₆₀₀ of each culture was centrifuged, then resuspended in 1 ml strict AP media (0.4% glucose, 20 mM NH₄Cl, 1.6 mM MgSO₄, 50 mM KCl, 20 mM NaCl, 120 mM triethanolamine, pH 7.4) plus carbenicillin (50 μg/ml). The mixtures were then immediately placed at −20° C. overnight. After thawing, 1 drop toluene was added to 1 ml of thawed culture. After vortexing, the mixtures were transferred to 16×125 mm test tubes and aerated on a wheel at 37° C. for 1 hr. 40 μl of each toluene treated culture was then added to 1 ml 1 M Tris-HCl pH 8 plus 1 mM PNPP (disodium 4-nitrophenyl phosphate hexahydrate) and left at room temperature for 1 hr. The reactions were stopped by adding 100 ml 1 M sodium phosphate pH 6.5. The OD₄₁₀ was measured within 30 minutes. Enzyme activity was calculated as micromoles of p-nitrophenol liberated per minute per one OD₆₀₀ equivalent of cells.

The results are summarized in Table 1.

TABLE 1 Determination of TIR Relative Strength: Use of PhoA as a Reporter Gene PhoA Standard Relative TIR Activity¹ Deviation Strength pBR322 0.0279 0.0069 — pPho51² 0.0858 0.0165 1 pSTBKPhoA#86 0.1125 0.0246 1 pSTBKPhoA#107 0.1510 0.0267 2 pPho41³ 0.1986 0.0556 3 pSTBKPhoA#81 0.2796 0.0813 4 pPho21⁴ 0.4174 0.1145 7 pSTBKPhoA#116 0.5314 0.1478 9 pPho31⁵ 0.5396 0.0869 9 pST116Pho 0.7760 0.1272 13  ¹micromoles of p-nitrophenol/min/O.D.₆₀₀ cells ²same STII variant as pSTIILys ³same STII variant as pSTIIBK#131 ⁴same STII variant as pSTIIC ⁵wild-type STII + MluI site, last codon GCC.

III. Secretion of Heterologous Polypeptide Examples

The plasmids used in these examples were all very similar in design as described above. Rather than describe in detail each construction, the expression plasmids are described here in general terms. Although a different polypeptide of interest was expressed in each example, the only significant variation between these constructions was the nucleotide sequence following the 3′ end of each coding region. Thus, for descriptive purposes, these plasmids were loosely grouped into the following two categories based on their 3′ sequence:

Category A: Within about 25 bp 3′ to the termination codon of each gene of interest began the sequence encoding the transcriptional terminator described by Scholtissek and Grosse (Nucleic Acids Res.15(7):3185 (1987)) followed by the tetracycline resistance gene of pBR322 (Sutcliffe, Cold Spring Harb Symp Quant Biol 43:77-90 (1978)). Examples in this category included plasmids designed for the secretion of mature NGF (Ullrich et al., Nature 303:821-825 (1983)), mature TGF-β1 (Derynck et al., Nature 316:701-705 (1985)) and domains 1 and 2 of ICAM-1 (Staunton et al., Cell 52:925-933 (1988)). A schematic representation of these plasmids is given in FIG. 17.

Category B: Examples in this category included plasmids designed for the secretion of mature VEGF (Leung et al., Science 246:1306-1309 (1989)), mature NT3 (Jones et al., Proc. Natl. Acad. Sci. U.S.A.87:8060-8064 (1990), RANTES (Schall et al., J Immunol 141(3):1018-1025 (1988)), and PhoA. The termination codon in each of these plasmids is followed in the 3′ direction by a segment of untranslated DNA (VEGF: approximately 43 bp; mature NT3: approximately 134 bp; RANTES: approximately 7 bp; PhoA: approximately 142 bp). Following this 3′ untranslated region, the sequence of pBR322 was re-initiated beginning with either the HindIII site (as in the mature NT3 secretion plasmid) or the BamHI site (PhoA, VEGF, RANTES secretion plasmids). A schematic representation of the plasmids included in this category is illustrated in FIG. 18.

These plasmids were used to transform the host E.coli strain 27C7. Transformant colonies were inoculated into 3-5 ml LB +carbenicillin (50 μg/ml). The cultures were grown at 37° C. with shaking for 3-8 hours. The cultures were then diluted 1:100 into 3 ml low phosphate medium (Chang et al., supra) and grown for about 20 hours with shaking at 37° C. For each culture, an 0.5 OD₆₀₀ aliquot was centrifuged in a microfuge tube.

Each 0.5 OD₆₀₀ pellet was then prepared for gel analysis as follows. Each pellet was resuspended in 50 μl TE (10 mM Tris pH7.6, 1 mM EDTA). After the addition of 10 μl 10% SDS, 5 μl reducing agent (1M dithiothreitol or 1M β-mercaptoethanol), the samples were heated at about 90° C. for 2 minutes and then vortexed. Samples were allowed to cool to room temperature, after which 500 μl acetone was added.

The samples were vortexed and then left at room temperature for about 15 minutes. Samples were centrifuged for 5 minutes. The supernatants were discarded, and the pellets resuspended in 20 μl water, 5 μl reducing agent, 25 μl NOVEX 2X sample buffer. Samples were heated at about 90° C. for 3-5 minutes, then vortexed. After centrifugation for 5 minutes, supernatants were transferred to clean tubes and the pellets discarded. 5-10 μl of each sample was loaded onto 10 well, 1.0 mm NOVEX manufactured gel (San Diego, Calif.) and electrophoresed for 1.5-2 hr at 120 volts. Gels were stained with Coomassie blue to visualize polypeptide (FIGS. 19-21).

To provide further quantitation of the results, some gels were analyzed by densitometry. These are displayed in Table 2 below. Both the polypeptide gels and the densitometry results indicated that the heterologous polypeptides tested were consistently secreted more efficiently when an STII variant of reduced translational strength was used to direct secretion of that polypeptide.

TABLE 2 Examples of Improved Polypeptide Secretion By TIR Modification: Densitometer Scans of Polypeptide Gels TIR Amount Secreted (Relative (% total host Polypeptide Strength) polypeptide) VEGF 9 0.6 3 5.9 NGF 9 1.6 7 1.8 4 5.7 1 5.5 RANTES 9 0.3 9 0.2 7 0.4 4 3.9 3 3.6 2 3.5  1* 1.6 1 1.7 TGF-β1 7 1.7 3 9.2 *pSTBKPhoA#86 signal sequence

23 88 bases nucleic acid single linear unknown 1 GCATGTCTAG AATTATGAAR AARAAYATHG CNTTYCTNCT NGCNTCNATG 50 TTYGTNTTYT CNATHGCTAC AAACGCGTAT GCCACTCT 88 36 bases nucleic acid single linear unknown 2 TTCAGCACCG CACAGAGTGG CATACGCGTT TGTAGC 36 82 bases nucleic acid single linear unknown 3 CTAGAATTAT GAAAAAGAAT ATCGCATTTC TTCTTGCATC TATGTTCGTT 50 TTTTCTATTG CTACAAACGC GTATGCCACT CT 82 75 bases nucleic acid single linear unknown 4 GTGGCATACG CGTTTGTAGC AATAGAAAAA ACGAACATAG ATGCAAGAAG 50 AAATGCGATA TTCTTTTTCA TAATT 75 67 bases nucleic acid single linear unknown 5 CTAGAATTAT GAAGAAGAAT ATCGCATTTC TTCTTGCATC TATGTTCGTT 50 TTTTCTATTG CTACAAA 67 67 bases nucleic acid single linear unknown 6 CGCGTTTGTA GCAATAGAAA AAACGAACAT AGATGCAAGA AGAAATGCGA 50 TATTCTTCTT CATAATT 67 79 bases nucleic acid single linear unknown 7 CGCGTATGCC CGGACACCAG AAATGCCTGT TCTGGAAAAC CGGGCTGCTC 50 AGGGCGATAT TACTGCACCC GGCGGTGCT 79 71 bases nucleic acid single linear unknown 8 CCGCCGGGTG CAGTAATATC GCCCTGAGCA GCCCGGTTTT CCAGAACAGG 50 CATTTCTGGT GTCCGGGCAT A 71 83 bases nucleic acid single linear unknown 9 GCATGTCTAG AATTATGAAR AARAAYATHG CNTTTCTTCT TGCATCTATG 50 TTCGTTTTTT CTATTGCTAC AAACGCGTAT GCC 83 27 bases nucleic acid single linear unknown 10 AGTGGCATAC GCGTTTGTAG CAATAGA 27 79 bases nucleic acid single linear unknown 11 CTAGAGGTTG AGGTGATTTT ATGAAAAAAA ACATCGCATT TCTTCTTGCA 50 TCTATGTTCG TTTTTTCTAT TGCTACAAA 79 79 bases nucleic acid single linear unknown 12 CGCGTTTGTA GCAATAGAAA AAACGAACAT AGATGCAAGA AGAAATGCGA 50 TGTTTTTTTT CATAAAATCA CCTCAACCT 79 506 bases nucleic acid double linear unknown 13 GAATTCAACT TCTCCATACT TTGGATAAGG AAATACAGAC ATGAAAAATC 50 TCATTGCTGA GTTGTTATTT AAGCTTGCCC AAAAAGAAGA AGAGTCGAAT 100 GAACTGTGTG CGCAGGTAGA AGCTTTGGAG ATTATCGTCA CTGCAATGCT 150 TCGCAATATG GCGCAAAATG ACCAACAGCG GTTGATTGAT CAGGTAGAGG 200 GGGCGCTGTA CGAGGTAAAG CCCGATGCCA GCATTCCTGA CGACGATACG 250 GAGCTGCTGC GCGATTACGT AAAGAAGTTA TTGAAGCATC CTCGTCAGTA 300 AAAAGTTAAT CTTTTCAACA GCTGTCATAA AGTTGTCACG GCCGAGACTT 350 ATAGTCGCTT TGTTTTTATT TTTTAATGTA TTTGTAACTA GTACGCAAGT 400 TCACGTAAAA AGGGTATCTA GAGGTTGAGG TGATTTTATG AAAAAGAATA 450 TCGCATTTCT TCTTGCATCT ATGTTCGTTT TTTCTATTGC TACAAATGCC 500 TATGCA 506 23 amino acids amino acid linear unknown 14 Met Lys Lys Asn Ile Ala Phe Leu Leu Ala Ser Met Phe Val Phe 1 5 10 15 Ser Ile Ala Thr Asn Ala Tyr Ala 20 23 90 bases nucleic acid double linear unknown 15 TCTAGAGGTT GAGGTGATTT TATGAAAAAG AATATCGCAT TTCTTCTTGC 50 ATCTATGTTC GTTTTTTCTA TTGCTACAAA YGCSTATGCM 90 78 bases nucleic acid double linear unknown 16 TCTAGAATTA TGAAAAAGAA TATCGCATTT CTTCTTGCAT CTATGTTCGT 50 TTTTTCTATT GCTACAAACG CGTATGCM 78 78 bases nucleic acid double linear unknown 17 TCTAGAATTA TGAAGAAGAA TATTGCGTTC CTACTTGCCT CTATGTTTGT 50 CTTTTCTATA GCTACAAACG CGTATGCM 78 78 bases nucleic acid double linear unknown 18 TCTAGAATTA TGAAGAAGAA TATCGCATTT CTTCTTGCAT CTATGTTCGT 50 TTTTTCTATT GCTACAAACG CGTATGCM 78 78 bases nucleic acid double linear unknown 19 TCTAGAATTA TGAAAAAAAA CATCGCATTT CTTCTTGCAT CTATGTTCGT 50 TTTTTCTATT GCTACAAACG CGTATGCM 78 78 bases nucleic acid double linear unknown 20 TCTAGAATTA TGAAAAAAAA CATTGCCTTT CTTCTTGCAT CTATGTTCGT 50 TTTTTCTATT GCTACAAACG CGTATGCM 78 78 bases nucleic acid double linear unknown 21 TCTAGAATTA TGAAGAAAAA CATCGCTTTT CTTCTTGCAT CTATGTTCGT 50 TTTTTCTATT GCTACAAACG CGTATGCM 78 78 bases nucleic acid double linear unknown 22 TCTAGAATTA TGAAAAAGAA CATAGCGTTT CTTCTTGCAT CTATGTTCGT 50 TTTTTCTATT GCTACAAACG CGTATGCM 78 90 bases nucleic acid double linear unknown 23 TCTAGAGGTT GAGGTGATTT TATGAAAAAA AACATCGCAT TTCTTCTTGC 50 ATCTATGTTC GTTTTTTCTA TTGCTACAAA CGCGTATGCM 90 

What is claimed is:
 1. A method of optimizing secretion of a heterologous polypeptide of interest in a cell comprising comparing the levels of expression of the polypeptide under control of a set of nucleic acid variants of a translation initiation region, wherein the set of variants represents a range of translational strengths, and determining the optimal translational strength for production of mature polypeptide, wherein the optimal translational strength is less than the translational strength of the wild-type translation initiation region.
 2. The method of claim 1, wherein the variants comprise nucleic acid variants of a secretion signal sequence.
 3. The method of claim 2, wherein the variant secretion signal sequences are variants of STII.
 4. The method of claim 3, wherein the STII variants are the following variants: 5′-TCTAGAGGTTGAGGTGATTTT ATG AAA AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCS TAT GCM 3′; (SEQ ID NO: 15, wherein Y at position 81 is C) 5′-TCTAGAGGTTGAGGTGATTTT ATG AAA AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAY GCG TAT GCM 3′; (SEQ ID NO: 15, wherein S at position 84 is G) 5′-TCTAGAGGTTGAGGTGATTTT ATG AAA AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAY GCS TAT GCC 3′; (SEQ ID NO:15, wherein M at position 90 is C) 5′ TCTAGAATT ATG AAA AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3′ (SEQ ID NO:16); 5′ TCTAGAATT ATG AAG AAG AAT ATT GCG TTC CTA CTT GCC TCT ATG TTT GTC TTT TCT ATA GCT ACA AAC GCG TAT GCM 3′ (SEQ ID NO:17); 5′ TCTAGAATT ATG AAG AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3′ (SEQ ID NO:18); 5′ TCTAGAATT ATG AAA AAA AAC ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3′ (SEQ ID NO:19); 5′ TCTAGAATT ATG AAA AAA AAC ATT GCC TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3′ (SEQ ID NO:20); 5′ TCTAGAATT ATG AAG AAA AAC ATC GCT TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3′ (SEQ ID NO:21); 5′ TCTAGAATT ATG AAA AAG AAC ATA GCG TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3′ (SEQ ID NO:22); and 5′ TCTAGAGGTTGAGGTGATTTT ATG AAA AAA AAC ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3′ (SEQ ID NO:23). 